Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells

Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs

The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins

Protein nonadditive expression and solubility contribute to heterosis in Arabidopsis hybrids and allotetraploids

Hybrid vigor or heterosis has been widely applied in agriculture and extensively studied using genetic and gene expression approaches. However, the biochemical mechanism underlying heterosis remains elusive. One theory suggests that a decrease in protein aggregation may occur in hybrids due to the presence of protein variants between parental alleles, but it has not been experimentally tested. Here, we report comparative analysis of soluble and insoluble proteomes in Arabidopsis intraspecific and interspecific hybrids or allotetraploids formed between A. thaliana and A. arenosa. Both allotetraploids and intraspecific hybrids displayed nonadditive expression (unequal to the sum of the two parents) of the proteins, most of which were involved in biotic and abiotic stress responses. In the allotetraploids, homoeolog-expression bias was not observed among all proteins examined but accounted for 17-20% of the nonadditively expressed proteins, consistent with the transcriptome results. Among expression-biased homoeologs, there were more A. thaliana-biased than A. arenosa-biased homoeologs. Analysis of the insoluble and soluble proteomes revealed more soluble proteins in the hybrids than their parents but not in the allotetraploids. Most proteins in ribosomal biosynthesis and in the thylakoid lumen, membrane, and stroma were in the soluble fractions, indicating a role of protein stability in photosynthetic activities for promoting growth. Thus, nonadditive expression of stress-responsive proteins and increased solubility of photosynthetic proteins may contribute to heterosis in Arabidopsis hybrids and allotetraploids and possibly hybrid crops

Caprin Controls Follicle Stem Cell Fate in the Drosophila Ovary

Adult stem cells must balance self-renewal and differentiation for tissue homeostasis. The Drosophila ovary has provided a wealth of information about the extrinsic niche signals and intrinsic molecular processes required to ensure appropriate germline stem cell renewal and differentiation. The factors controlling behavior of the more recently identified follicle stem cells of the ovary are less well-understood but equally important for fertility. Here we report that translational regulators play a critical role in controlling these cells. Specifically, the translational regulator Caprin (Capr) is required in the follicle stem cell lineage to ensure maintenance of this stem cell population and proper encapsulation of developing germ cells by follicle stem cell progeny. In addition, reduction of one copy of the gene fmr1, encoding the translational regulator Fragile X Mental Retardation Protein, exacerbates the Capr encapsulation phenotype, suggesting Capr and fmr1 are regulating a common process. Caprin was previously characterized in vertebrates as Cytoplasmic Activation/Proliferation-Associated Protein. Significantly, we find that loss of Caprin alters the dynamics of the cell cycle, and we present evidence that misregulation of CycB contributes to the disruption in behavior of follicle stem cell progeny. Our findings support the idea that translational regulators may provide a conserved mechanism for oversight of developmentally critical cell cycles such as those in stem cell populations

Characterization of egg chamber and stalk defects in <i>Capr</i>- ovaries.

<p>A-C) Germarium, budding egg chamber, and stalk (white bracket) of the indicated genotypes stained with antibodies to the follicle cell marker, FASIII (green), and with TO-PRO-3 iodide (red). A) <i>Df(3L)Cat</i>/+ (Control), B) <i>Capr-</i> showing a reduced primary stalk, and an aberrantly packaging egg chamber displaying an absence of stalk (dashed bracket) and misencapsulation of a single germline cell (arrow), and C) <i>Capr-</i> containing a disorganized stalk. D-E) Egg chambers stained for TO-PRO-3 (red). Optical sectioning revealed 16 nuclei in the control egg chamber (D), but fewer cells in a <i>Capr-</i> egg chamber (E) including nuclei of inappropriate size for this stage (arrowhead). Scale bar is 30 microns.</p

Loss of <i>Capr</i> disrupts germline cyst development.

<p>A) Schematic of the <i>Drosophila</i> germarium with bars below indicating the numbered germarium regions (1, 2a, 2b, 3) and their cell types: non-proliferating terminal filament (TF) and cap cells (CC), germline stem cells (GSC) which give rise to the developing 16-cell cysts (cyst), escort cells (EC) which facilitate movement of cysts through regions 1 and 2a, and the follicle stem cells (FSC) which give rise to the follicle cells (FC) and stalk. Anterior is to the left in all figures. B-C) Immunofluorescence analysis of control <i>+/Df(3L)Cat</i> (B, B’) or <i>Capr²/Df(3L)Cat</i> (C, C’) germaria stained with antibodies to Slit (green and B’, C’) and TO-PRO-3 iodide (DNA, red). Arrowheads indicate the position of FSCs and the unencapsulated cysts are numbered in B’ and C’. Scale bar is 30 microns.</p

Loss of <i>Capr</i> alters cell cycle dynamics but not proliferation rates in the FSC lineage.

<p>A-C) Fixed <i>Df(3L)Cat/+</i> (control), or <i>Df(3L)Cat/Capr²</i> (<i>Capr-</i>) germaria were stained with antibodies to phospho-histone H3 (red), and FasIII (green), and with TO-PRO-3 iodide (blue). A) Quantification of the % of germaria scored that showed any phospho-histone H3-positive staining in cells of the FSC lineage. The number of germaria analysed was 81 control, 82 <i>Capr</i>-. B-C) Examples of stained germaria of the indicated genotypes. Size bar is 30 microns. D) Quantification of the % of pulse labeled germaria scored that incorporated BrdU in cells of the FSC lineage. The difference between <i>Df(3L)Cat/+</i> (control), or <i>Df(3L)Cat/Capr²</i> (<i>Capr-</i>) was not significant (P  =  0.09). The number of germaria analysed was 80 control, 86 <i>Capr</i>-. E) Tangential section of a fixed stage-10 egg chamber stained for GFP (green) and FasIII (red). The <i>Capr-</i> follicle cell clone (no GFP staining) and its adjacent wild-type twin-spot (bright green) are of similar size. Size bar is 60 microns.</p

Loss of <i>Capr</i> increases the number of unencapsulated germline cysts.

<p>The percent of total germaria scored (n) containing the normal number of Slit-stained 16-cell cysts (≤5 cysts), or supernumerary 16-cell cysts (>5 cysts), is shown for each genotype. <i>Df</i> refers to the <i>Capr</i> deficiency, <i>Df(3L)Cat</i>.</p

Loss of <i>Capr</i> leads to loss of follicle stem cells but not germline stem cells.

<p>The heat shock-FLP system was used to generate clones homozygous for the FRT80B (control) or for the FRT80B <i>Capr²</i> (<i>Capr²</i>) chromosome. Percent germaria containing follicle stem cell clones (A), or germline stem cell clones (B) were quantified at 9 and 15 days after heat shock (AHS). The number of germaria analysed for 9 day and 15 day data respectively was 106 and 166 for control and 78 and 166 for <i>Capr²</i>.</p